1. Field of the Invention
Present-day gene transfer techniques allow the possibility for the development of more productive crop plants at a pace much faster than before. Gene transfer and selection of the best plants has been the objective of breeding programs for years. Powerful new techniques for gene transfer recently have been developed for moving single genes and whole blocks of genes from one plant to another and even for moving genes from non-plants into plants. These new techniques can help mankind preserve the genetic diversity of plants by actually creating greater diversity through gene recombination.
This invention relates to a novel method for producing transformed germplasm which circumvents many of the limitations associated with the present-day technology in this field.
2. Description of the Prior Art
Several methods have been developed for the transformation of eucaryotic cells of plants and animals by foreign DNA. These methods include protoplast fusion [W. Schaffner, Proc. Nat'l. Acad. Sci. U.S.A. 77: 2163-2167 (1980); M. Rassoulzadegan et al., Nature 295: 257-259 (1982)], DEAE dextran [J. H. McCutchan et al., J. Nat'l. Cancer Inst. 41: 351-357 (1968)], calcium phosphate coprecipitation with DNA or recombinant bacteriophage [F. L. Graham et al., Virology 52: 456-467 (1973); M. Ishiura et al., Mol. Cell. Biol. 2: 607-616 (1982)]. More recently, electroporation has been successful in introducing DNA into plant protoplasts [M. Fromm et al., Proc. Nat'l. Acad. Sci. U.S.A. 82: 5824-5828 (1985); M. Fromm et al., Nature 319: 791-793 (1986)], fibroblasts [H. Liang et al., Biotechniques 6: 550-558 (1988)], and mammalian rod blood cells [T. Y. Tsong et al., Biblio. Haematol. 51: 108-114 (1985); G. Chu et al., Nucl. Acids Res. 15: 1311-1326 (1987)]. The success of the electroporation method is dependent, in part, on optimizing parameters relative to the membrane, the DNA, and the electric field. Evidence for the success of transformation after electroporation has been measured by incorporation of radioactively labeled DNA [Tsong et al., supra], transient gene expression [H. Potter et al., Proc. Nat'l. Acad. Sci. U.S.A. 81: 7161-7165 (1984); O. Smithies et al., Nature 317: 230-234 (1985)], and the formation of stable transformants [C. D. Riggs et al., Proc. Nat'l. Acad. Sci. U.S.A. 83: 5602-5606 (1986); H. Stopper et al., Z. Naturforsch. 40c: 929-932 (1985)].
There are several drawbacks to current methods of DNA transformation in plants. One of these is that the transfer of DNA usually involves the use of isolated protoplasts. Protoplasts from many plant species are recalcitrant to regeneration into mature genetically stable plants and many of the most important economic crop plants have never been regenerated from protoplasts. In most cases where regeneration has been achieved, the production of plants from transformed protoplasts involves lengthy sterile culture and regeneration procedures. Further, many DNA transformation procedures involve the use of Agrobacterium tumefaciens, and its tumor-inducing (Ti) plasmid. The number of plant species that are infected by this system is extremely limited, and other vectors are not currently available.